As loss of cell cycle regulation is a hallmark of carcinogenesis, identifying novel therapeutic targets aimed at restoring growth control is an important approach to cancer therapy. Endometrial cancer (ECA) is the most common gynecologic malignancy in the US with 49,470 new cases and over 8,190 deaths estimated for 2013. The major objectives of this proposal address mechanisms involved in the pathogenesis of estrogen (E2)- induced Type I ECA (85% of ECAs) and a new potential therapy for this disease that involves inhibiting degradation of the tumor suppressor, p27kip1 (p27), a key cell cycle protein that arrests cell proliferation. We reported that E2 induces ubiquitin-mediated degradation of nuclear p27 by its specific E3 ligase, SCF- Skp2/Cks1, in endometrial epithelial (EECs) and ECA cells with increased cell proliferation. Conversely, progesterone (Pg; a therapy for low grade ECA) as well as TGF-, increase p27 in the nucleus and inhibit cell proliferation by preventing p27 degradation. Therefore, we provide compelling evidence that p27 is a key target for growth regulation in the endometrium and in endometrial carcinogenesis. We have identified novel small molecule inhibitors of Skp2/Cks1 (Skp2E3LIs) that only increase nuclear p27, which is critical since cytoplasmic p27 mediated migration/metastasis. Furthermore, Skp2E3LIs block both E2-induced degradation of nuclear p27 and proliferation in vitro in ECA cells and {in vivo in mouse EECs}. Importantly, Skp2E3LIs have the potential to be a major therapeutic advancement over current general proteasome inhibitors that indiscriminately block protein degradation including oncogenes. Whereas Skp2/Cks1 causes p27 degradation, the E3 ligase APC/Cdh1 targets Cks1/Skp2 for destruction leaving p27 intact. Interestingly, we show that E2 decreases Cdh1and thereby increases Skp2/Cks1 for p27 degradation whereas Pg and TGF- increase Cdh1 to increase nuclear p27. Therefore, p27 degradation can be inhibited both, by increasing Cdh1 or by blocking Skp2. As such, we will test two main hypotheses: 1. that the Cdh1-Skp2/Cks1-p27axis is important in cell cycle dysregulation by the ubiquitin proteasome system (UPS); 2. that Skp2E3LIs have significant translational value for the treatment of ECA. We propose to: 1. Use E2, Pg, and TGF- as molecular switches to learn how Cdh1 is controlled to affect p27 levels. 2. Show that co-localization of p27 and Cdh1 in human biopsy tissue is a biomarker for positive outcomes of Pg therapy. {3. Perform co-crystallization/NMR structural analysis of Skp2-Cks1-Skp2E3LI complexes for chemically optimizing current lead compounds to define the role of p27 in ECA in vitro and in vivo.} 4. Quantify the effects of Skp2E3LIs on intracellular [nuclear] trafficking of p27 Skp2, and Cks1 together with cell cycle analysis by single cell imaging microscopy. 5. Test Skp2E3LIs for their efficacy in blocking the growth of human ECA tumors in mouse models. As the degradation of nuclear p27 occurs in numerous cancers, our studies should impact the cancer biology field by providing mechanistic insights into the role of the UPS in cancer and the use of Skp2E3LIs as a novel approach to cancer therapy.